Structure-Function of AAV - a Viral Gene Therapy Vector

描述（由申请方提供）：腺相关病毒（AAV）是人类基因治疗的主要候选载体。长期目标是理解衣壳-宿主相互作用和病毒组装的结构和机制基础。这需要对衣壳进行工程改造，以最大限度地发挥AAV将治疗性DNA递送至患有癌症或遗传性疾病的靶细胞的潜力。绘制细胞受体结合位点的足迹对于改变组织向性至关重要，而绘制中和抗原决定簇对于工程化可以到达先前暴露于AAV的患者中的靶细胞的病毒是必需的。其他病毒载体也将面临类似的挑战，这项工作将成为一个范例。 这项研究将建立在我们最近的3埃分辨率的AAV血清型2（AAV-2）的晶体结构，并继续完善和分析。与细胞受体硫酸乙酰肝素蛋白聚糖的相互作用将通过与小乙酰肝素片段复合的AAV-2的晶体学分析、通过与较大片段复合的冷冻电子显微镜（EM）（与Ken Taylor合作）以及通过结合位点的定点诱变来表征。 将使用由巴里卡特和Jurgen Kleinschmidt制备的单克隆抗体（MAb）组研究AAV-抗体相互作用。功能性表位将通过在抗体存在下通过病毒增殖选择的突变体的测序来定位。代表性MAb的物理表位将通过冷冻EM成像进行定位，使用已知的AAV-2结构以分子分辨率进行解释。 将启动其他4种血清型中至少一种的结构研究。比较分析将显示受体结合区域的保守程度，并揭示最受免疫监视的区域的可变性程度。所有拟议的结构研究将补充和加速其他地方开发基于AAV的疗法的广泛努力，为目前正在通过开明的试错法进行的修改提供结构原理和一系列限制性约束。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) is a prime candidate vector for human gene therapy. The long term objective is an understanding of the structural and mechanistic bases of capsid-host interactions and viral assembly. This is needed to engineer modifications of the capsid to maximize AAV's potential to deliver therapeutic DNA to targeted cells afflicted with cancer or an inherited disorder. Mapping the footprint of the cellular receptor binding site is critical to modifying tissue tropism, while mapping of the neutralizing antigenic determinants is needed to engineer viruses that can reach target cells in patients previously exposed to AAV. Similar challenges will be faced with other viral vectors, for which this work will be a paradigm. The research will build upon our recent 3 Angstrom resolution crystallographic structure of AAV serotype 2 (AAV-2), and its continuing refinement and analysis. Interactions with the cellular receptor, heparan sulfate proteoglycan, will be characterized through crystallographic analysis of AAV-2 complexed with small heparan fragments, through cryo-electron microscopy (EM) of complexes with larger fragments (in collaboration with Ken Taylor), and through site-directed mutagenesis of the binding site. AAV-antibody interactions will be studied using panels of monoclonal antibodies (MAb) prepared by Barrie Carter and Jurgen Kleinschmidt. Functional epitopes will be mapped through the sequencing of mutants to be selected by viral propagation in the presence of antibodies. Physical epitopes for representative MAb will be mapped by cryo-EM-imaging, interpreted at molecular resolution using the known AAV-2 structure. Structural studies of at least one of the other 4 serotypes will be initiated. Comparative analysis will show the extent of conservation of receptor-binding regions, and reveal the extent of variability of regions most subject to immune surveillance. All of the proposed structural studies will complement and accelerate extensive efforts elsewhere to develop AAV-based therapies, by providing a structural rationale and set of limiting constraints for modifications that currently are being made by enlightened trial-and-error.